Chromatography procedures include affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. The use of affinity chromatography for the purification of biological macromolecules is well-established. Under optimal conditions in the laboratory, the results of affinity chromatography purifications can be spectacular. For example, yields of 90% and purification factors of over 6000 have been reported for avidin, Cuatracasas, P. and Wilchek, M., Biochem. Biophys. Res. Commun. 33, 235 (1968), and vitamin B.sub.12 -binding protein, Allen, R. H., and Majerus, P. W., J. Biol. Chem. 247, 7702 (1972). The success of affinity chromatography in small-scale purifications has been harder to realize on a larger scale partly because of the expense and difficulty involved in chemically derivatizing solid supports with affinity ligands. In addition, it is difficult to achieve high flow rates with these packings, and they are not easy to sterilize and still maintain their biological activity. For this reason, there is a continued interest in the development of more convenient methods of attaching specific ligands to solid supports.
The use of heterobifunctional ligands in affinity chromatography has recently been proposed. See Mattiasson, B. and Olsson, U., J. Chromatog. 370, 21 (1986); Olsson, U. and Mattiasson, B., J. Chromatog. 370, 29 (1986). In this work, trypsin was connected to a sepharose support by a conventional cyanogen bromide reaction. A heterobifunctional ligand comprising dextran, with soybean trypsin inhibitor (STI) and Cibacron Blue substituted thereon, was prepared, and the heterobifunctional ligand bound to the sepharose through the interaction of trypsin and STI. The Cibacron Blue so bound to the sepharose was used to extract lactate dehydrogenase from bovine heart extract. While this procedure permits Cibacron Blue to be removed from the sepharose for sanitizing of the sepharose and replacement with a different ligand, the procedure disadvantageously employs a potentially toxic cyanogen bromide reaction, and requires the binding of a protein, trypsin, to the solid support. The protein is subject to denaturation and microbial attack.
In view of the foregoing, a first object of the present invention is to provide a chromatography apparatus in which chromatographic functional groups are easily and reversibly bound to a solid support.
A second object of the present invention is to increase the capacity of the solid support for the chromatographic functional groups bound thereto.
A third object of the present invention is to provide new means for associating chromatographic functional groups with the groups which reversibly bind the chromatographic functional groups to the solid support.
A fourth object of the present invention is to provide a way to reduce nonspecific binding to the solid support in a chromatography apparatus.
A fifth object of the present invention is to provide a method of making chromatography apparatus in which the chromatographic functional groups are reversibly bound to the solid support, and a sixth object of the present invention is to provide a means for removing chromatographic functional groups reversibly bound to the solid support.
A seventh object of the present invention is to provide different materials as the solid support in chromatography apparatus which achieve some or all of the foregoing objects.
An eighth object of the present invention is to provide ion exchange chromatography apparatus which achieve, at least in part, the foregoing objects.
A ninth object of the present invention is to provide hydrophobic chromatography apparatus which achieve, at least in part, the foregoing objects.
A tenth object of the present invention is to provide materials which can be used to produce chromatography apparatus as described above, and particularly materials which can be used to produce chromatography apparatus in which nonspecific binding to the solid support is reduced.
The foregoing objects are achieved by the apparatus, methods, and materials disclosed below.